JPH06510024A - Pharmaceutical preparations and methods - Google Patents

Abstract

A process for preparing a liposomal cyclosporin therapeutic formulation, which comprises dissolving a combination of a neutral and negatively charged phospholipid and a cyclosporin in an organic solvent; drying the solution to form a solid phase, and hydrating the solid phase in an aqueous solution having a pH ranging from 7.5 to 9.5.

Description

[Detailed description of the invention] Pharmaceutical preparations and methods The present invention relates to the fields of biochemistry and medicine, and in particular to novel liposomal preparations and and methods. More particularly, the present invention relates to liposomes containing the immunosuppressant cyclosporine. Concerning soum preparations and their manufacturing methods. The present invention also provides liposol with reduced toxicity. Concerning muciclosporin preparations.

Cyclosporin was discovered in 1970 by researchers in an attempt to identify a new fungicide. It was done. Cyclosporine (also known as cyclosporine A), competent immune The inhibitor is two strains of Deuteromycetes, Cylindrocapon Lucidum B. ooth and Tolypocladium inflatum from Gam5 Separated.

Cyclosporins are hydrophobic, neutral compounds with essentially similar chemical and physical properties. It is a cyclic peptide. Cyclosporin is a typical example and all of the 1201 It consists of 7 amino acids with a molecular weight. Cyclosporin is used for methanol and chloroform. soluble in water and ether and essentially insoluble in water. This is a proprietary Intravenous preparations or Labrophil and ori dissolved in mustard oil and alcohol It is used therapeutically as either an oral product dissolved in oil.

Tucrosvorin primarily treats allograph patients and has been used in experiments against autoimmune diseases. This drug is 1 Since its appearance in 978, it has greatly improved the survival rate of transplant patients.

Cyclosporine is a very useful immunosuppressant, but it also Extremely useful when used both for long periods of time and at high doses needed to ensure It is extremely toxic. The most severe side effect associated with cyclosporine treatment is drug-induced kidney disease. It is toxic. Vascular interstitial toxicity is the most common and simplest form of cyclosporine nephrotoxicity. - or as three different morphological disorders occurring in combination.

These morphological changes associated with cyclosporine nephrotoxicity may be associated with cyclosporine. Although not specific to toxicity, this may be observed in combination with other If there is a correspondingly high level of serum cyclosporine, the disorder is probably due to cyclosporine. This is a result of crosporin toxicity. Some people have whole blood of 200-500 ng/ml and Therapeutic doses (5 to 1 On+ (g/kg/day) indicates these harmful effects. Nephrotoxicity is creatiny The increase in drug levels is followed by serological monitoring. This at creatinine level The increase is likely a direct result of arterial constriction and occlusion, which results in a lower renal system filtration rate and therefore less blood flow. This will result in an increase in serum creatinine.

There are other negative side effects associated with cyclosporine treatment.

This occurs with different frequencies depending on the type of implantation. This is associated with cardiovascular hypertension and spasticity. , dermatophytosis, gingival hyperplasia, diarrhea, nausea, vomiting, liver toxicity, leukopenia, and phosphorus Hematopoietic degeneration, including lymphoma, dyspnea, and signs of sinusitis.

Another side effect associated with intravenous transfer of tucrosvorine is the intravenous carrier vehicle, C. It is based on remophor-E L (Cr e L). CreL is Polymer is one of the best ionic surfactants used to dissolve lipophilic drugs. It is lyoxyethylated castor oil. The most common adverse effects associated with CreL administration The most common is hypersensitivity, which results from the rapid release of histamine and increases cause high blood pressure. Is this also part of the nephrotoxicity associated with cyclosporine treatment? This is thought to be enhanced by CreL deposition and crystal formation within the renal tract. Other studies Both renal blood flow and creatinine clearance decreased in animals treated with (:reL). was shown. Liconic acid, a component of CreL, has been shown to cause vasoconstriction; This is also linked to high blood pressure and decreased glomerular blood flow.

Formulating drugs in liposomes for the purpose of administration, thus toxic linseed oil No attempt has been made to eliminate the toxicity of tucrosvorin by eliminating the vehicle. It is.

Liposomes form closed, fluid-filled spheres when mixed with water; It is a microscopic transport compound made from lipids. Phospholipid molecules are polar and parent It has an aqueous ionizable head and a hydrophobic tail consisting of a long fatty acid chain. Therefore, ten The tail is optionally attached to exclude water when a large number of phospholipid molecules are present with water. while the hydrophilic phosphny 1-head interacts with water. This result is a two-layer film. , where the fatty acid tails converge within the newly formed membrane and the polar head Orient in the opposite direction towards the aqueous medium. On one surface of the membrane, the polar head is an aqueous liposome. Direct towards the inside. On the opposite surface, the polar head interacts with the surrounding aqueous medium . As liposomes form, water-soluble molecules are incorporated into the aqueous interior and Lipophilic molecules show a tendency to be incorporated into lipid bilayers. Liposomes contain many lipids Bilamellar, like an onion, with liquid separating the two layers, or entirely liquid. Either unilamellar with a single bilayer surrounding the center.

Many types of liposome manufacturing techniques that can be used and yield different types of liposomes There is. Applications, chemicals intended to entrap, and to form bilayer films It can be selected depending on the type of lipid used.

Other parameters that must be considered when producing optimal liposome preparations are: similar to that of the regulated release mechanism. This is as below: ( 1) high percentage of chemical entrapment: (2) increased chemical stability, ( 3) low chemical toxicity: (4) rapid method of manufacture, and (5) reproducible size distribution. .

The first method described for encapsulating chemicals in liposomes is This included the production of MLVs. This MLV method involves dissolving lipid components in an appropriate solvent. evaporation of the solvent and aqueous medium to form a dry lipid film. including lipid hydration. The bilamellar vesicles that are formed generally have three or more concentric 2 It is a structure with multiple layers. Add lipophilic drugs to MLVs by co-dissolving drugs in the solvent layer while the hydrophilic drug is entrapped between the bilayers with a hydration buffer. . By increasing the length of hydration time and shaking the resuspended lipid film slows down. This results in a high proportion of aqueous phase per mole of lipid, thus facilitating hydrophilic drug encapsulation. It can be improved. The increased entrapment of aqueous buffers also increases the charged Obtained by using lipids.

Liposomes also have diameters up to 2 μm, but are generally smaller than 1 μm. It can be formed as lamellar vesicles (UVs).

There are several techniques used to generate unilamellar liposomes. Reverse phase evaporation By using this method, large unilamellar vesicles can be formed. this is pressure Remove the organic layer of the emulsion under sonication of phospholipids, buffer and excess existing solvent. It is done by This technique is particularly useful for hydrophilic molecules such as ferritin, 2 Encapsulate a large volume aqueous phase containing 5S RNA or 5V-40 DNA It is useful for The maximum encapsulation (65%) of the LUV aqueous layer is Obtained when the on-strength is low (0.01M NaCl): the ionic strength is 0.5 Encapsulation decreases to 20% with increasing M NaCl. Dimensions of LUVs The method varies depending on the fat and cholesterol content. l:1 molar ratio of cholesterol and phosphorus Vesicles formed from lipids are 0.47μ based on entrapped volume. Heterogeneous vesicles with an average diameter of m and a size range of 0.17–0.8 μm Form a size distribution. prepared from a similar phospholipid mixture lacking cholesterol Vesicles have an average size of 0.18 μm and a size range of 0.1-0.26 μm. Ru.

Solvent injection evaporation methods produce both large and small UVs, depending on changes in technology. be able to. Phospholipids to diethyl ether to form large UVs 55-65° containing the substance to be dissolved and entrapped or injected Inject into buffer maintained at C. The mixture is kept under vacuum at 30°C. solvent When it evaporates, vesicles are formed. The diameter range of these vesicles is 0 ．． It is 25 to Iμm. This process is well suited for entrapment of large molecules. are doing.

Small unilamellar vesicles can also be formed using a variety of techniques. ethanol By dissolving phospholipids in arbitrarily reorganizes into unilamellar vesicles. This was produced by ultrasonication produce UVs with an internal volume (0.2-0.5 L1 mol/lipid) similar to Provides an easy way to Sonication or extrusion of MLVs through a filter ) produces a dispersion of UVs with a diameter of up to 12 μm, which is transparent or semi-transparent. Occurs as a clear suspension.

Another common method of producing small UVs is detergent removal techniques. Io phospholipids ionic or non-ionic detergents, e.g. cholates, triton Soluble in either X or n-alkyl glucoside. Then the lipid in which the drug is dissolved - Mix with detergent micelles. Then some techniques: dialysis, gel filtration, affinity -Remove the detergent by one of the following methods: chromatography, centrifugation, or ultrafiltration. This person The size distribution and entrapment efficiency of UVs produced by the method depend on the technology used. Depends on the details. Also, when entrapping proteins, the detergent is The uncertainty that, when removed, the protein restores to its native, active conformation It has an umbrella.

Therapeutic uses of liposomes include the delivery of drugs that are normally toxic in their free form. child In liposomal form, toxic drugs are directed away from sensitive tissues and delivered to selected areas. Target. Liposomes also release drugs gradually over a long period of time It can be used therapeutically to reduce the frequency of administration. Furthermore, liposomes are static A method of forming an aqueous dispersion of a hydrophobic drug for intravenous transport can be provided.

Liposomes are encapsulated against selected host tissues and away from sensitive tissues. When used to target chemical agents, several techniques can be used. Ru.

These steps manipulate the dimensions of the liposome, its net surface charge as well as the route of administration. including doing. More specific manipulations have receptors or antibodies in special locations within the body. labeling the liposomes.

The route of liposomal delivery can also influence distribution throughout the body. Passive transfer of liposomes Delivery includes the use of various routes of administration, such as intravenous, subcutaneous and topical. Each route is This results in differences in the localization of posomes. Activate liposomes to selected target areas Two common methods have been used to target the surface of liposomes with antibodies or specific The goal is to bind any of the receptor ligands. Antibodies are highly specific for their corresponding antigens It is known for its ability to bind to the surface of liposomes, thus making liposomes It has been shown that target specificity of encapsulated drugs can be increased.

Liposomes are suitable for intravenous delivery, as the chemical composition of many drugs precludes intravenous administration. very useful for adapting drugs to Many hydrophobic substances including cyclosporine Sex drugs cannot be easily dissolved in water-based media and cause toxic reactions in living organisms. This category applies because it must be dissolved in an alcohol or surfactant that has been shown to Enter Chuu. liposomes consisting primarily of lipids, with or without cholesterol Mums are non-toxic. Furthermore, since liposomes are made from amphiphilic molecules, this It can entrap hydrophilic drugs in its internal space and hydrophobic molecules in the lipid bilayer. .

To entrap enough cyclosporine into a stable liposome carrier Therapeutically effective techniques may not be available for various reasons, primarily due to the incompetence of those skilled in the art. Rosporin-inserted liposome products are not commercially available. (with a high proportion of activator) formulations that can be incorporated internally and are sufficiently stable for commercial use. There was a special need to develop liposomal cyclosporine containing.

The present invention provides such a product.

Summary of the invention a physiologically compatible buffer, preferably between 7.0 and 9.5, preferably between 7.5 and 7.5; Using an aqueous solution with a pH of 9.1, negatively charged phospholipids and cyclospores were Preparing stable immunosuppressive formulations by hydrating a solid lipid phase containing phosphorus . This solid lipid phase is created by dissolving phospholipids and cyclosporin in an organic solvent. Can be formed. Neutral phospholipids, such as phosphatidylcholine, preferably 16 having a lipid moiety essentially consisting of an 18-carbon fatty acid straight chain, or contained in this solid lipid phase. The negatively charged phospholipid is preferably a phosphatide. ruglycerol, preferably consisting essentially of straight chains of 14 to 18 carbon fatty acids or phosphatinok acids, such as distearoylphosphatide. Fatidylglycerol, dipalmitoylphosphatidylglycerol, Simiri Stoyl phosphatidylglycerol or simyristoylphosphatisic acid It is.

More particularly, liposomal cyclosporine therapy is stable even when not lyophilized. Prepare the preparation for use by a method consisting of the following steps: (a) (i) phosphatidylcholine, (ii) phosphatidylglycerol, phosphatidylcholine, a compound selected from the group consisting of sphatisic acid and mixtures thereof; and (i ii) Dissolve cyclosporin in an organic solvent to prepare the model of (1) vs. (11) vs. (iii). forming a solution with a ratio ranging from about 7:7:0.05 to about 7:3:2; (b) The organic solution thus formed is dried to form a solid fat, e.g. a film or form a powder; (C) Hydrate and stabilize this solid fat with an aqueous solution having a pl of about 7.5 to about 9.5 form a liposomal cyclosporine therapeutic preparation.

In another aspect, the invention provides for aqueous solutions having a pH ranging from about 7.5 to about 9.1. Suspended negatively charged lipids and cyclosporins, preferably cyclosporins A therapeutic lipid composition comprising:

In a preferred embodiment of the invention, the liposomes are monolabeled with a diameter of 0.2 μm or less. They are Meller vesicles. Furthermore, cholesterol is preferably phosphatidyl coli. Can be incorporated into liposomes in amounts up to 50 mol% of Add.

The present invention provides novel method and storage stable and therapeutically effective amounts of active ingredients. provides a cyclosporine-inserted liposome formulation with reduced toxicity. A posomal cyclosporine formulation is provided. The method of the present invention A commercially viable process for the production of phosphorus is provided.

Brief description of the drawing Figure 1 is a graph showing the results of a test used to measure liposome formulations. be.

Figure 2 shows the results of tests used to determine the concentrations of various components of liposomal formulations. This is a graph showing the results.

Figure 3 shows the test used to determine the molar ratio of DMPG in liposome formulations. This is a graph showing the results.

Detailed description of the invention The term liposome as used herein refers to U.S. Pat. ．． Unilamellar vesicles or bilamellar vesicles as described in No. 935.171 the contents of which are inserted here as a reference. Terms used here Packellation refers to the incorporation of cyclosporine within the liposome membrane.

The method of the invention begins with the preparation of a solution in which liposomes are formed. In summary, Certain amounts of phosphatidylcholine, phosphatidylglycerol and cyclosporin The solution is dissolved in a solvent, preferably chloroform, to form a solution. For example, use a rotary evaporator, spray dryer or other equipment to evaporate the film or powder. form a solid lipid phase such as This film is then heated in a range of about 7.0 to about 9.5 The liposomal dispersion is formed by hydration in an aqueous solution having a pH ranging from

Preferably a thin film evaporator as described in U.S. Pat. No. 4,935,171. It is used to form multiple melar ribonomes by stirring the dispersion. For example, super Sonication or homogenization such as a Gauljn homogenizer or French breath Monolamellar labeling is achieved by application of shear forces to the aqueous dispersion of the lipid solid phase by the use of a Form a sicle. Ether injection, freezing and thawing, dialysis of detergent solutions from lipids, shear force using or other known methods used to prepare liposomes. can also be applied. Liposolysis can be performed using various known techniques, including continuous shear force. The dimensions of the room can be adjusted. Preferably as described in U.S. Pat. No. 4,753,788. 7000 to 13.0 using a modified Gaulin homogenizer apparatus. 00 psi pressure and the approximate aggregation of lipids) and transition temperature of 200 nanometers. It forms unilamellar vesicles with a diameter of less than 100 ml. Multilamellar or single lamellar Methods of agitation or shearing lipids to form melar vesicles are well known to those skilled in the art. known and not as such part of the present invention.

Distearoyl phosphatidylcholine is egg phosphatidylcholine (egg PC) A preferred phosphatidylcholine for use in the invention. Preferred phosphatidylco Phosphorus may be obtained from soybeans or other plant materials or may be partially or wholly synthetic. including, for example, dipalmitoylphosphatidylcholine. All of these are city It's on sale.

Phosphatidylglycerol suitable for use in the present invention is distearoyl phosphatide. Dilglycerol (DSPG) and Simyristoylphosphatidylglycerol (DMPG). Other phosphatidylglycerols suitable for use include Contains urylphosphatidylglycerol (DLPG). these things is commercially available.

Instead of or in addition to this phosphatidylglycerol, similystoyl phosphatide Sfatinoic acid, distearoylphosphatic acid, and dipalmitoylphosphatic acid Sphatisic acid can be used. This ingredient is about 7:7:0.05 to about 7:32 , most preferably ranging from 7:5:2 to phosphatidylcholine. The molar ratio of cyclosporine to cyclosporin or phosphatic acid preferred in solution. Or mix it. Other molar ratios such as 7:5:I to 7:3:l are also substantially equivalent. As a result, it can be used.

The preferred organic solvent is chloroform, but ether, methanol, ethanol and Other solvents or combinations of solvents such as alcohols and other alcohols can also be used.

Suitable aqueous solutions for hydration purposes include buffered solutions, e.g. phosphate buffer (0,145M NaC1,0, 003M NaH2PO4,0, 0035M NaH2PO4) (P BS).

The formulation prepared according to the above method contains a high percentage of cyclosporin, 60 % or more, and is stable on storage, which indicates that this formulation make the therapeutic use of the product commercially viable. This formulation may also be lyophilized and stored. can be rehydrated without loss of efficacy. Dose tube for free cyclosporine The principles are well known to medical practitioners and are useful for suppressing immune responses in mammals and especially humans. The amount of cyclosporin-inserted liposome formulations of the invention that is effective will be apparent to those skilled in the art. That's it.

Encapsulation of cyclosporin in the manner described reduces the toxicity of existing therapeutic preparations. and enhance the effectiveness of the treatment.

The invention will be more fully understood with reference to the following examples.

This is intended to be illustrative of the invention and not limiting. Example 1? Example 6 describes the formation of liposomal cyclosporine of the present invention and the chemical and biological reagents. A series of liposome formulations containing tucrosvorin using rotary evaporation technology Prepare a product in which lipids and cyclosporin are dissolved in an organic solvent (chloroform). The solution thus formed was then dried on a rotary evaporator. Then this dry fill The membrane was hydrated in an aqueous solution buffered to a pH of 6.5 to 9.1 to form bilamellar vesicles. was formed. cyclosporin powder (M, W, = 1201) in a 7:5:2 molar ratio, Similis J. Ilphosphatidylglycerol (DMPG).

M, W, = 684) and egg phosphatidylcholine (M.

W, = 786) to cyclosporin multilamellar vesicles (cyclosporin-ML Vs) was prepared. The raw material solution of cyclosporine, DMPG and egg PC was prepared in lum. Cyclosporin raw solution 1.20 in a 100ml round bottom flask ml, egg PC raw material solution 2.75 ml and DMPG raw material solution 1. ．． 70m1 set Cyclosporin-MLVs was prepared by combining. Next 95r Place this round bottom flask in a rotary evaporator with a water bath temperature of 36 °C set at pm. The organic solvent chloroform was allowed to evaporate by standing.

3.0ml of sterile 0.01M PBC with pH 7.8 heated to 52°C was mixed with drug-lipid. Add to the round bottom flask containing the film and heat to 52°C! -1, it was heated Cyclospoly when placed in a shaker and rotated at 120 rpm for 20 minutes. -MLVs were formed. Next, add a small sterile magnetic stirrer to the round bottom flask. Heat this on a hot plate for about 5-10 minutes at low heat (60°C). Remove the film by stirring vigorously (high setting) helped. The cyclosporine-MLVs were transferred to sterile tubes. pH 7,8 Separately add bacterial PBS to a 2.0 ml round bottom flask and add the remaining MLVs to the flask. washed.

Centrifuge the pooled MLVs at 2987xg for 20 minutes and remove the supernatant. The MLV pellet was then suspended in sterile PBS, pH 7, 8, 5, 0 ml.

MLVs were centrifuged at 2987xg for 20 minutes. After removing the supernatant, the final ML The V pellet was resuspended in sterile PBS, pH 7, 8, 5, 0 ml.

Other than deletion of cyclosporine, described for cyclosporine-MLVs Empty-MLVs at the same time as cyclosporin-MLVs using the same process as (E-MLVS) was prepared. In this process, liposomes were formed with the following ingredients: .

#l DMPG Cyclosporin 10:1 molar ratio #2 Egg PC: Ncrosbori 10.1 molar ratio #3 Egg PC rainbow crosporin 50:1 molar ratio liposome Each of the samples was diluted 1=30 with methanol and used spectrophotometric analysis, 44 , 900 to obtain the optical density (0,D,) at 214 nm. The concentration of cyclosporine was measured.

The results are shown in Figure 1, where DMPG, cyclosporine, and a liposomal formulation containing cyclosporine 0.57 mg/ml; It can be seen that it is significantly higher than that formulated with egg PC.

Example 2 Another series of bilamellar vesicle samples, namely liposomes, as described in Example 1. Prepared according to the method. These samples were as follows: #l Egg PC: DMPG Nijicrosborin 7:5.1 molar ratio #2 Egg PC: DMPG Nidicrosborin 3:31 molar ratio #3 Egg PC: DMPG/Cyclosporin 5.31 molar ratio The MLV sample was diluted 1:30 with methanol and spectrophotometric analysis was used. , 0.0 at 214 nm using an extinction coefficient of 44.900. By obtaining D, The concentration of crosporin was measured.

The results of spectrophotometric analysis are shown in Figure 2, where the ratio of 7:51 is egg PC: DMPG. MLVs containing rainbow cyclosporine capped with 0.63 mg/ml cyclosporine. The concentration of DMPG is significantly higher than that of the same composition at other molar ratios. Another series of bilamellar vesicles was prepared according to the method described in Example 1 at different degrees. Prepared. The samples were as follows.

#1 Egg PC: DMPG Nidicrosborin 75:1 molar ratio #2 Egg PC: DMPG Nidicrosborin 7:7:1 molar ratio #2 Egg PC: DMPG/Cyclosporin 731 molar ratio #4 Egg PC: DMPG/Tucrosborin 7:l:1 molar ratio This sample was diluted to 130% with methanol and cyclosporin was added as in the previous example. The concentration of ion was measured. The spectrophotometric results are shown in Figure 3, where the 7:5:1 egg PC:　DMPG　M　L　V　with lower molar ratio of crosborin or significantly higher amounts of cyclosporine than MLVS containing DMPG. (0.99 mg/ml).

Example 4 Another series of MLVs was prepared according to the method described in Example 1. This At Leeds, the amount of cyclosporine was varied as follows: #l Egg PC: DMPG Nidicrosborin 7.5:1 molar ratio #2 Egg PC: DMPG Cyclosporin 7:5:2 molar ratio #3 Egg PC: DMPG Nidicrosborin 7.5 to 0.5 molar ratio The above sample was diluted l:30 with methanol and using spectrophotometric analysis, 4 cyclo by obtaining O,D at 214 nm using an extinction coefficient of 4.900. The concentration of sporin was measured.

The results of this analysis are shown in Table 1. As can be seen, encapsulated cyclosporine The amounts were essentially the same at all three molar levels.

Table 1 Lipid encapsulated with various molar ratios of cyclosporine: 0. D. All Nokuro times Contained encapsulated drug ratio @214nml #phosphorus Tsucrosborin percentage 7:5:0.5 0.36 0.6mg 0.26mg 43%7:5:l O, 781.2mg 0.57mg 47% 7:5:2 1.38 2.4m g 1.0] mg 42 01 Another series of liposomes according to the method described in Example 1 Somes were prepared. In this series, all liposomes (7:5221 The flPc:DMPG Nijikrosborimbolin ratio was used. However, hydration The buffer pH was varied between values of 6.5 and 9.1.

This sample was then diluted to 130 with methanol and an extinction coefficient of 44.900 was used. The concentration of cyclosporine was then measured again by spectrophotometric analysis at 2141 m.

The results are shown in Table 2, where the best buffer pH in terms of % drug recovery was 7.8. However, it has been found that satisfactory results can be obtained at pH values ranging from 7.2 to 9.1. Ru.

Table 2 Effect of pH on encapsulation efficiency pH6, 57, 27, 57, 88, 29, 1% tucrosborin Encapsulation 28.7 66.0 71.7 88.0 77.0 -72.3 (Initial drug collection) % standard deviation 5.5 3.0 2.9 6.0 3.0 2.0 as described in the example above From the results, the molar ratio of egg PC:DMPC was 7:5/0.05 to 7:5:2. Liposomal formulations containing diclosborin are more stable during the formation process than conventional formulations. It is clear that encapsulating Rosporin is much more effective.

For optimal results, hydration should be at a pH of about 7.0 to about 9.5, preferably 7.8. It is essential that the process be carried out in a medium that has

The stability of the formulations of the invention is illustrated by the following examples.

Example 6 When cyclosporine-MLVs were freshly prepared or rehydrated following lyophilization, Cyclosporine MLV binding for various times (2 hours, 24 hours, 4 hours) 8 hours) to demonstrate the effect of different temperatures (4°C, 125°C and 37°C). experiment was used. 7:5:2 egg PC:DM using the rotary evaporation technique described above. Prepare cyclosporine insertion MLVs with molar ratio of PG rainbow cyclosborine. Ta.

The hydration buffer was PBS, pH 7,8. Following hydration, MLV 050-80 Sephadex column in a 5n+1 syringe. and centrifuged at 250 rpm for 10 minutes. This is tucrosvorin inserted MLV This was done to separate unencapsulated cyclosporin from s.

Following centrifugation, the cyclosporine-MLV pellet was reconstituted in PBS, pH 7,8. Suspended and pooled to yield a total volume of approximately 9 ml. This pooled trial The samples were measured for tucrosvorin concentration by ultraviolet absorption spectrophotometry.

The total sample volume was divided into two portions of 4.5 ml each. Add one part to three more aliquots (1.5 ml per aliquot). Other parts for at least 2 hours It was frozen to 70°C and then lyophilized.

Unfrozen aliquots (1.5 ml each) containing fresh cyclosporine-MLVs ) were incubated at either 4°C, 125°C (room temperature) or 37°C, and Samples (0.5 ml) were taken from each temperature condition at 2 hours, 24 hours and 48 hours. . Remove and pass each sample through a Sephadex G50-80 column using a 5 ml syringe. Centrifuge and resuspend the cyclosporine-MLV pellet. It was analyzed for the concentration of

Prepare lyophilized cyclosporine-MLVs by rehydrating them with sufficient sterile deionized water. The product was returned to its original capacity. This rehydrated material was divided into cyclosporine concentrations. and then divided into three 1.5 ml aliquots, which were incubated at 4°C and 125° C (room temperature) or 37°C. Sample (0.5ml) was heated for 2 hours, 24 hours and Sephadex G in a 5 ml syringe was removed from each storage state after 48 hours. Centrifuge through a 50-80 column and pellet each cyclosporine-MLV. were resuspended and analyzed for cyclosporine concentration. The results are shown in Tables 3 and 4. Summarize in table.

New cyclosporine-MLVs Storage time concentration initial chemical Temperature (mg/ml) % of 111 degrees 4°C2h O, 5580% 4°C24h O, 5681χ 4°C48h O, 5681% 25℃　OO,69 25°C2h O, 4565]; 25°C 24h O, 4870% 25°C 48h O, 4870% 37°C00,69 37℃　2h　O, 4565% 37°C 24h 0.48 70% 37℃ 48h O, 2333% Table 4 Lyophilized but rehydrated cyclosporin-MLVs storage time concentration First medicine Temperature (mg/ml) % of concentration 4℃ 2h O, 5085% 4℃ 24h O, 4983% 4℃ 48h O, 4373% 25°C00,59 25°C2h O, 4475% 25℃ 24h O, 4576% 25℃ 48h O, 4475% 37°COO, 59 37°C2h O, 4373% 37℃ 24h 0.40 68% 37℃ 48h 0.26 44% This data is based on freshly prepared and lyophilized, rehydrated cyclosporine-ML. The lyophilization and rehydration steps showed similar results for both MLV and Vs. s and cyclosporine binding is not significantly altered. Both types of preparations are tested There was some chemical loss after 2 hours of incubation at ambient temperature. death From Kashina, when the preparation was stored at 4°C for 2 hours, MLVs and cyclosporin (15-20% drug loss) at either 25°C or 37°C (25-20% chemical loss). (35% chemical loss).

Prepared a cyclosporine-MLV formulation that is stable and exhibits optimal drug encapsulation. By preparing various lipids, their molar ratios are determined by several factors, including the pH of the hydration buffer. I checked. Cyclosporine is a hydrophobic molecule and is released from the aqueous space within liposomes. Rather, it is incorporated into the lipid membrane, so it is best suited to maximize the lipid capacity per vesicle. We selected multilamellar vesicles for our first experiment.

Initial studies were performed using cyclosporin and other lipids. DMPG d Crosborin MLVs is significantly higher than egg PC Nijicrosborin MLVs. showed good encapsulation. This is its transition temperature, approximately 23°C (room temperature). This is due to the flexibility of PG, and this is due to the presence of cyclosporin in the liposome bilayer. It is possible to adapt large molecules such as molecules. I used non-hydrogenated egg PC. The reason for this is that unlike synthetic lipids, egg PC contains one or more lipids, each with a different chain length. This is because the liposome bilayer contains a large amount of cyclosporin. Also helps to adapt the child.

Liposomal hecyclosporosis using various combinations of lipids, egg PC and DMPG In optimizing the phosphorus formulation and minimizing its leakage from MLVs, egg PC: A molar ratio of DMPG Nidiclosporin of 7:5:I showed the highest amount of drug encapsulation. did. This suggests that the properties of both lipids in the right ratio may contribute to increased encapsulation efficiency. It suggests that.

To further investigate the binding of negatively charged DMPG and cyclosporin in the liposome membrane. For this purpose, researchers varied the hydration buffer pl for cyclosporine liposome binding. The effectiveness was tested. Maximum encapsulation was observed in 0.15M PBS, pH 7.8 and modifying the pH above or below this value will further change the encapsulation efficiency. 6.5 Using a hydration buffer with a pl between 25 and 9.5 at most 88 yielding drug recoveries ranging in the range of %.

Using increasing amounts of cyclosporine in liposomal formulations to reduce the lipid-to-drug ratio. The final formulation study was conducted using These studies have shown that the molar ratio of cyclosporine 2-fold increase (7:5:2 molar ratio, egg PC: DMPG rainbow crosborin) There was no change in the percentage of the initial drug recovered in the somes. This is less fat A large amount of cyclosporin-MLVs is produced in quality, and a certain amount of encapsulated drug is produced. This is important because it reduces the cost and time required to produce the results.

Tested to determine the therapeutic efficacy of cyclosporine liposomal formulations of the invention I did this.

Materials and methods are described in the section below and testing is detailed in Examples 7-12.

Preparation of drug formulation for treatment of mice Cr with 0.1 ml of 95% ethanol emophor-eL 1. Dissolve 12 mg of cyclosporin powder in Oml. and then free cyclosporin (cyclosporin) by heating to 50°C. Phosphorus-CreL) was prepared. After cyclosporine has dissolved, heat to 55°C Sterile 0.15M PBS.

Add 7, 8, 1, 9 ml of pH to the solution to produce a Schiff 0 sporin concentration of 4 mg/ml. It was. The PBS used had the following composition: 0.0028M monosodium phosphate O, 0.0072M disodium phosphate and O, 145M sodium chloride. Change This solution was then diluted with PBS to produce a final cyclosporine concentration of 2.0 mg/ml. did. 2.0 mg/ml cyclosporine solution 1, sterile 0.15M P to 0 ml Add BS, PH 7, 8, 9, 0 ml to cyclosporine concentration of 0.2 mg/ml will occur. Since the doses required for the mice are different, we next test three different concentration series. Prepare the crosporin solution and similar the volume of injected material for all treatments. (0.1 to 0.2 ml).

Cyclosporine-MLVs and E as listed in the second paragraph of Example 1 MLVs were prepared. Mix the chemicals with methanol and tetrahydrofuran (THF), v/v, 1: When dissolved in I, the Beers-Lambert equation and 238r+ In Examples 7-12, by using the absorbance for cyclosporin at m The concentration of cyclosporine was measured in the sporin-MLV suspension. Anhydrous methanol Add 0.1 ml of cyclosporine-MLV sample to a test tube containing 1.4 ml. Cyclosporin was extracted from Cyclosporin-MLVs by child Open the test tube for about 10 seconds, add 1.5 ml of THF, and add to the solution. It created a vortex again. E-MLV samples were treated similarly. This extraction process is cyclo Make a 1=30 dilution of the Sporin-MLV sample and analyze spectrophotometrically at 238 nm. and compared with a 1:30 dilution of the E-MLV sample. Cyclosporine concentration was measured Later, 1 to 2 ml aliquots of cyclosporine were placed in a Revco freezer. Gradually frozen to 0°C. When the sample is completely frozen, freeze-dry it and freeze it. Stored at 20°C. Before use, resuspend it in sterile distilled water and incubate for 10 minutes. ℃ and reanalyzed for cyclosporine concentration.

If necessary, dilute this sample with sterile PBS, pH 7,8 for injection. A suitable concentration was obtained.

Animal model for cyclosporine treatment Mice used in this experiment were C57BL. /6J, female mice, 8 to 16 weeks old. At different times, depending on treatment management, Mice were treated with various doses of cyclosporine-MLVs and cyclosporine-CreL. were inoculated intravenously. Sterilize the blood by cardiac puncture or retroorbital exsanguination into the victim's mouth. I took it out. Serum was collected and later hemagglutination analysis and blood urea nitrogen (BUN) Stored at -20°C for measurements. Remove some kidneys and add 0.15M PBS. Fix in diluted 1096 formalin solution, pH 7.3 and section. Stored at 4°C. The spleen was also removed aseptically and homogenized using a sterile tissue mofosonizer. It became. The spleen homozygotes from each treatment group were pooled and RPMI 1640 6. Neutrop in a 15 ml sterile conical centrifuge tube. hil l5olation Medium 4. Layered on Oml. this The material was centrifuged at 225×g for 30 minutes at 18-20°C. isolated lymph Take out the sphere band from N1M Gradient and RPMI 6. Dilute with Oml Ta. Centrifuge the cells in this zone at 125Xg for 9 minutes, discard the supernatant, and remove the pellet. RPMI the cut 6. Resuspend in Oml and centrifuge again at 125 x g for 9 min. did. Discard the supernatant again and transfer the final pellet to RPMI 1. Resuspend in Oml I let it happen.

Cells stained with 0.1% Trypan blue using a hematocytometer counting chamber. Cell concentration and viability of this final pellet was determined by counting cells. this Concentrations were adjusted to complete RPMI (10% newborn calf serum, 2% penicillin-streptomycin). RPMT with syn and glutamine) to 5 x 106 cells/ro11.

Then for modified Jerne plaque analysis and T-lymphocyte embryogenesis analysis. This cell suspension was used.

Kennedy analysis modified Jerne plaque analysis guinea pig complement 15 dilutions of this complement were prepared by diluting with magnesium saline: 5XIO’ by diluting the cell suspension 1:10 in saline solution. A lymphocyte suspension was prepared. Complement l5 dilution 1.0ml and 296sRBC1 , 1% sheep red blood cells containing a 110 dilution of complement by mixing Oml (sRBC) suspension was prepared.

Kennedy assay (Kennedy 1971) was performed in a 96-well flat bottom tissue culture dish. Each linoleum sample from a given treatment group was tested in triplicate. each well Add 0.1 ml of lymphocyte suspension (5x lO' cells) in complete RPMI tissue medium. cells/mi), 0.05 ml of 1% 5RBc with 1:10 complement and complete R,P 1 ml of MIO was added. Some control wells contain 0.1 ml of lymphocyte suspension. , 1%5RBc0.05ml without conbriment and complete RPMI O,1 It contained ml. One well contains 0.05ml of 1% 5RBc and complete RPM1 Mix 2 ml of O, and in another well 0.2 ml of RPM1 1:10. Two separate controls by mixing 0.05 ml of 1% 5RBc with complement Wells were prepared. The culture dish is then placed in a humid environment (lined with a damp paper towel). The cells were placed in an incubator at 4°C for 1.25 hours. Next, this culture Place the feeding dish at room temperature for 20 minutes and ink at 37°C with 1596 CO2/95% air ink. The cells were cultured in an incubator for 1.5 hours. Following the final incubation, 400 Determine plaques by counting plaques in each well with a x inverted microscope. Formation was assessed and the number of plaques averaged over three wells for each sample.

Results are reported as plaque forming units (PFU)/10' cells.

T-lymphocyte PHA stimulation and chemotaxis analysis 96-well embryonic development of T-IJ lymphocytes. Round bottom tissue culture dishes were performed. 5XI06 cells prepared from each treatment group in each well /ml lymphocyte suspension (isolated for Kennedy analysis) 0.1ml added. Cell suspension samples from each treatment group were run in duplicate. Each of the test wells 900% of phytohemagglutinin (PHA) diluted with RPMI 1640. ug/ml ml solution 0.1 and l complete RPMI 1640.0.05 ml added. Control wells contained 0.1 ml of lymphocyte suspension and 15 ml of complete RPMI O. Contained. Next, place the tissue culture dish in a 37°C incubator with 5% CO2/95% air. The cells were cultured for 60 hours. After a 37°C incubation period, the material from each well was Pipette into a microfuge tube and incubate for 10 minutes 83 Centrifugation was performed at 20×g. Transfer each supernatant from this centrifugation to a separate microfuge. Pipette into tubes and freeze at -20°C until use in chemotaxis analysis. I tied it up and kept it.

Microscope slides were acid washed with 3M HCl and 95% ethanol in an I:1 ratio for 2 hours; Negative charges were reduced by rinsing with deionized water and soaking in 0.5% gelatin for 5 minutes. . The slides were finally washed with distilled water and allowed to air dry.

1. agarose in 50 ml of distilled water. Dissolve Og and 0.25 g of gelatin Agarose slides were prepared by the following method. Add this preparation to au 1~clay. cooled to 47℃ and mixed with warmed RPMI 1640.50ml. Approximately 3 ml of this solution was then pipetted onto each glass slide. Agarose is After solidification, 3-well rows were created in the agarose for each slide.

Since macrophages are localized in the mixed lymphocyte zone of this gradient, the N1M separation technique Macrophages were extracted from spleen homogenates of non-chemically treated C57BL/6J mice using The page was separated. Next, transfer the lymphocyte/monocyte suspension to complete RPM1 1640 3X. The cells were adjusted to 107 cells/ml. Then three wells in agarose on each slide were filled as follows: 3X107 cells/m115μ in the center well! , outer u RPMI 15μ in well! and 15 μl of the cyst generation supernatant sample in the other outer well. 0 Place this slide in a sterile humidification chamber (a sterile petri dish lined with a moist sterile filter). (dish) and incubator at 37 °C with 5% CO2/95% air for 18 h. It was cultured in After incubation, soak in absolute alcohol for 30 minutes, then for 30 minutes. The slides were fixed by immersion in 37% formaldehyde, pH 7.3. The agarose was then carefully removed from the slide. Wri this slide Ght 5 stain and examine these with a micro projector. Macrophage migration was assessed by measuring cell migration from the edge of the well. .

Hemagglutination analysis of serum from 5RBC-stimulated mice. Serum samples obtained from each mouse were used. Make 2-fold dilutions (1:1 to 1:102) with 0.15M PBS, pH 7.3. 4) was prepared. The analysis was performed in a 96-well, round-bottom tissue culture dish. Each test 0.05 ml of 0.5% 5RBc (IXIO@cells/ml) and 0 serum samples in the wells. ．． Control wells contain 0.05ml of 0.5% 5RBc and O,15M Contained 0.05 ml of PBS, pH 7.3. Place this plate on a shaker It was then rotated at +00 rpm for 10 minutes, followed by incubation for 1 hour at room temperature. 40 Blood cells were determined for each sample by examining the wells under a 0x inverted microscope. The agglutination value was measured.

Hemagglutination analysis of serum from LPS-stimulated mice 5R bound to lipopolysaccharide (LPS) A BC suspension was prepared as described by Andersso (1971).

1.0 mg LPS from Salmonella abortus equi /ml in sterile 0.15M PBS, pH 7.3. Next, this L P.S. (3.0 ml,) was boiled in a glass screw cap tube for 2 hours. heating Afterwards, this LPS was cooled to 37°C in a water bath, and 1.0 ml of packed 5RBc was added to the LP. S was added, followed by a separate incubation for 45 minutes at 37°C.

Next, the 5RBC-LPS mixture was washed three times with 0.15M PBSS pH 7°3. and diluted to 1x107 cells/ml.

0.15M PBS, PI (7,3, 1:1 to 1:2048 for each sample) Serum samples were prepared for this analysis by performing two-fold dilutions. as mentioned above The analysis was performed with the exception that 5RBC-LPS suspension was used instead of 0.5% 5RBC. Using.

Histology of mouse kidneys following cyclosporine treatment at least buffered at 4 °C. After setting in formalin, the kidneys were further fixed as follows = 70% ether. 2 hours in ethanol, 1.5 hours in 80% ethanol: 1.5 hours in 90% ethanol 3 incubations of 1.5 h each in 100% ethanol; 2 ink hairs for 1.5 hours each and 3 inks of 1.5 hours each in paraffin. incubation. Kidneys were embedded in paraffin and 6 μm sections were obtained. child Sections were stained as follows: 2 immersions in dehydrating agent for 2.5 min each time, 1 immersion for 2 min each time. ．． 00% ethanol twice, 95% ethanol for 2 minutes, 70% ethanol for 2 minutes Wash with water, wash with acid-alcohol in madoxylin for 2.5 minutes, wash with ammonium water. Soaked 10 times, washed with water, stained with eosin for 1.5 minutes, immersed in 95% ethanol 10 times, twice in 100% ethanol and twice in dehydrating agent.

Nephrotoxicity was determined by examining sections with an Iff microscope at 100x and 400x. Renal sections were evaluated.

In vitro antibacterial effect of cyclosporin Three species of Aspergillus fumigatus, A. fumigatus, A. flavus and A.

niger and the yeast cryptoccus neoformans in this study. I used this part. These cultured bacteria are Ca1ifornia 5tatePo lytecnicυn1versity, Pomona, Microbiology Cultured Bacteria Collection and laboratory strains #385, #359, #360 respectively. and was named #608.

The medium for growing these organisms is 5abouraud (SAB) dex It was trose broth and SAB dextrose agar (1.5%). These training The four types of bacteria used in the study were maintained on SAB agar slants. Collect spores for testing Previously, Aspergillus aspergillus cultures were transferred every 48 hours for 6 days. Sterilization 0.85 Spores were collected by washing the slant with 1.5 ml of % saline. haematosa Count the spores using a cytometer counting chamber and quantify the number of spores in 0.85% saline. Xl0” spores/ml.C, Neoformance yeast cells were also transferred: however, using SAB broth The cells were collected using Yeast cells are counted using a hemacytometer counting chamber. The cells were counted and the number of cells was adjusted to 1X10' cells/ml in SAB broth.

To measure the antibacterial activity of cyclosporine-CreL and cyclosporine MLVS. For this purpose, SAB plates were inoculated with chemically treated spores (or yeast cells) as follows; Add 0.15 ml of 1×]0 cospores/ml suspension to an appropriate concentration of cyclosporine-ML. Vs or cyclosporine-CreL (1 μg, 5 μg, 10 μg or 200 μg g) in test tubes and in different tubes to obtain a final volume of 0.5 ml in each tube. Add enough SAB broth to each tube containing the drug concentration: then swirl all tubes. Then pour it onto another SAB plate and spread it evenly with two sterile curved glass rods; After 36 hours of incubation at 7°C, the colonies were counted and their diameters were determined. It was measured.

Encapsulation of cyclosporine within MLVs alters its antibacterial in vitro activity To determine whether the yeast Cryptococcus neoforman s and molds, Aspergillus 1 usflavus, fumigatus and Production of cyclosporine-MLVs and cyclosporine-CreL on B. niger A study was conducted to compare the in vitro antibacterial activity. Encapsulation (cyclosporine-MLV ) and unencapsulated (cyclosporine-CreL) after 36 hours of incubation. The number of C. neoformance colonies was inhibited in vitro after vation.

A concentration of 1 μg of cyclosporine inhibited yeast by 50% and cyclosporine- CreLIμg reduced the number by 65% compared to the control untreated sample. Cyclos Increasing porin-MLV or cyclosporine-CreLi (5 μg, 1 0gg and 200μg) compared to 10 colony counts for control untreated samples. 38, IO and 2 and cyclosporin against MLVs. There was a further decrease in colony counts of 3.3 and 0 for CreL. Cyclos Porins also affected colony size of C. neoformance. lμg Exposure of yeast to cyclosporine-CreL even at low concentrations There was an 87% reduction in size compared to treated samples. Cyclosporin-CreL A similar degree of inhibition of yeast colony size was observed when using 5μg or 10gg. No detectable yeast was found when yeast were challenged with 200 μg of the drug.

In contrast, yeast colony size decreased in the presence of various amounts of cyclosporine-MLVs. Inhibition of the method showed a dose response: l11g, 5μg, 10μg and 20L4/ , g the colony size was 33%, 67%, 80% and 939% compared to the control, respectively. There was.

In either form, tucrosborin reduces the number of Aspergillus spp. However, A, flavus, A, fumigatus and A, Inhibits niger colony size.

Cyclosporine-MLVs increased by 50% compared to control untreated cultures, 7896.9 A, fl at concentrations of 2% and 96% 1μg, 5μg, IOμg and 200μg avus colony size was reduced. However, cyclosporine-CreL Compared to control, 60% for 1μg%, 85% for 5μg, 10gg% and 96% for 200 μg and 98% for 200 μg, Cyclosporin-MLVs showed more significant inhibition of A.flavus.

Cyclosporin-MLVs has a concentration of In2 of A and fumigatus. At 10 and 200 μg, this drug did not inhibit colony size, but In comparison, 4096 and 96% of the bacteria were inhibited, respectively. In contrast, cyclosporine -CreL inhibited the colony size of A. fumigatus at 5 μg and higher doses of IOμg and 200μg, respectively, compared to untreated controls. The reductions were 30%, 80% and 96%.

A, niger colony size for cyclosporine-MLVs and cyclosporine -Cr was decreased by both eL.

Cyclosporine-NILVs at concentrations of 1 μg, 5 μg, to μg and 200 μg 3794.75%, 7596 and 97.596 bamboo shoots compared to control, respectively. Reduced Ronnie dimensions.

Cyclosporine-CreL was 1 μg, 5 μg and 10 μg, respectively, compared to untreated control. Since the colony size was reduced by 87.5%, 87.5% and 92,596 times, showed greater inhibition. At 200 μg, inhibition of bacterial growth was significant for both types of drug. It was comparable.

These data indicate that the encapsulation of cyclosporine in MLVs is 10 gg or less. The in vitro antibacterial efficacy of free drugs against certain bacteria when administered at doses below Indicates that the Higher drug levels (i.e. 200μ of any form of drug) g) shows comparable in vitro antibacterial activity compared to 7-crosporin-N (LVs and cyclosporin). To investigate whether porin-CreL has comparable immunosuppressive activity in vivo. In order to Treated with some form of medicine. The B cell activity of these mice against this antigen was Monitored by Jerne pull analysis and serum hemagglutination antibody titers. experimental In the first cent, mice were treated five times with various dose levels of the drug. Jern The results of e-plaque analysis were determined in mice challenged with 5RBC or not treated with tucrosborin. The cell has a plaque forming unit value (PFU) of 560/IO@cells. Indicated. However, when mice fed with 5 RBCs were treated with Empty- There was a 1.4-fold increase in PFU compared to untreated, antigen-challenged mice. 5 ．． treated with 5 doses of cyclosporine-CreL at 15 or 25 mg/kHz. Mice challenged with 5RBC showed 25%, 57% and 84% reduction compared to untreated controls, respectively. showed a decrease in plaque. ! , 5.15 or 25 mg/kg cyclospori Mice treated with MLVs were challenged with 5RBC and untreated controls, respectively. A reduction in plaques of 45%, 54%, 6896 and 89% was shown. 5 mg/ 5 doses of cyclosporine-M (total = 0.55 mg per mouse) Plaque levels in mice treated with LVs were 5 doses of 15 mg/kg. (total = 1.65 mg per mouse) of cyclosporine-Cre treated mice. It can be compared to that of Usu.

Mouse serum from the above experiment was analyzed for hemagglutination titer. Non-antigenic challenge, no Treated mice had hemagglutination titers of 0. Just 5RBC, 5RBC and CreL again Mice given 5RBC and empty -L had a titer of I:853. 5RB C and either 5 or 25 mg/kg cyclosporine-CreL. 80% and 99%, respectively, compared to mice given only 5 RBCs. 596 showed a decrease in antibody titer. 1.5, J5, or 25 mg/k Mice treated with cancer polyrin MLVs were compared to mice given only 5RBCs. 97.5% and 99.596.99.5% in antibody titer compared to and showed a 99.8% decrease. Thus, cyclosporine-MLVs were treated with compared to mice given a comparable dose of cyclosporine-CreL. had a lower antibody titer compared to the previous one. This result also applies to CreL treatment or empty -L We show that none of the treatments alter the antibody response of these mice to 5RBC. did.

With 5 doses of either cyclosporine-CreL or cyclosporine-MLVs T cells from treated mice were stimulated with PHA following their isolation. Rinhokai The ability to generate chemotaxis and chemotakines was measured by chemotaxis analysis and determined as the chemotaxin coefficient. It was expressed as Non-tucrosvorin-treated mice had a chemotaxin coefficient of 1.43. did. 5.15 or 25 mg/kg cyclosporine-Cre treatment Mice had chemotaxin coefficients of 39 stones, 3796 stones, and showed decreased chemotaquinone production as indicated by a 50% decrease in the 5.15 Or mice treated with 25 mg/kg cyclosporine-MLVs were compared to untreated mice. Chemotaquinones decreased by 3696.411% and 38.5%, respectively, compared to had a number. Therefore, simply giving 5 mg/kg tucrosporine-MLVs Mice treated with 15 mg/kg cyclosporine-Cre showed suppression of chemotaxin production comparable to that of .

The next set of experiments tested lower total doses of cyclosporine-CreL and cyclosporine-CreL. The immunosuppressive effect of Rosporin-MLVs was investigated. In these in vivo experiments , tuculosporin-MLVs or tuculosporin-CreL. Mice were treated with the following doses: Jerne plaque analysis results indicate that cyclosporine 5RBC-challenged mice without administration had a plaque level of 680 PFU/IO' cells. It was shown that Mice treated with 5RBC and CreL showed high levels of plaque. (700), but with 3 doses of 5 mg/kg (total = 0 per mouse). ．． 33 mg) or 15 mg/kg (total = 0.99 mg per mouse) 5RBC-challenged mice treated with Rosporin-Cre compared to non-cyclosporin-treated mice. Plars decreased by 50% (340) and 68(%) 220, respectively, compared to U.S. It had a crebel.

5 RBC and 3 doses of 5 mg/kg (total = 0.33 mg per mouse) or I 5 mg/kg (total = 0.99 mg per mouse) of cyclospor Mice treated with cyclosporine-MLVs received a comparable dose of cyclosporine-CreL. were further immunosuppressed in their 5RBC responses than mice treated with (respectively, 7 0% (200) and 79% (140) decreases).

When mouse serum from the above experiment was analyzed for hemagglutinating antibody titers, Jerne Results similar to those seen with plaque analysis were obtained as seen in Table 5.

Table 5 Drug (serum collected on the 90th day) Potency Gl/Control (sRBC only) I:8 53G2: 5. Omg/kg Tsucrosborin-CreL 2341G 3: 15.0 mg/kg cyclosporine-CreL 1: I28G4 : 5. Omg/kg Tucrosporine-MLVs 1:128G 5: 15. Omg/kg cyclosporine-MLVs l: 43 cases reported in 9 In the experiment, C57BL/6J mice were treated with 10% 5RBCIP on the 1st and 4th IE. o, 2 ml was inoculated. This mouse has Tuclosborin-CreL or Cyclospo Rin-! With 3 intravenous doses (day 0, day 4 and day 8) of 'vl LV　s Processed. Control mice G1 were inoculated with 5RBCs only. All mice are number 9 Hemagglutination analysis was performed on serum pooled from 5 mice/group sacrificed on a daily basis. Ta. Results were replicated three times with 5 mice/group and are reported as mean titers.

Mice receiving only 5RBC developed a titer of 18530, whereas 5RBC Treated with either BC and 5 or I 5 mg/kg cyclosporin-CreL compared to non-cyclosporine-treated mice, the titer was 2.5 times or 6 times higher, respectively. 5RBC and 5 or 15 mg/kg cyclosporine-Ni Mice treated with either LVs showed a 6.7-fold and 19.8-fold decrease, respectively. Ta. Comparable immunosuppression of 5 RBCs was 5 mg/kg cyclosporine-MLVs and was seen in mice administered 15 mg/kg cyclosporine-CreL. .

Tm cells from mice treated with cyclosporine three times were stimulated with PHA and chemostimulated. When tested for taxine production, the chemotactic coefficient is shown in Table 6. It can be calculated to measure sea urchin T cell activity.

Table 6 PHA-stimulated T cell chemotaxin production on day 8; serum collection on day 9) Experiment 1 Actual Test 2Gl: Control (no link a suphorin) 1.76 1.60G 2: CreL O, 15ml of 25% dilution 1.78 1.50G3: 5.0 mg/kg Tsuk0 Suphorin-CreL 1.50 1.19G4:1 5.0 mg/kg Tuclosporin-CJeL 1.17 1. αΩG5: 5.0 mg/kg Cycloshori 7-MLVS 1.07 1.00G6:1 5. Omg/kg Tsukurosupori 7-MLVS O, 890, reported in 78 cases 9 In an experiment, three doses (days 0, 4 and 8) of cyclosporine-Cre were administered. Treat C57BL/6J mice with either L or cyclosporine-M L Vs. I understood. Control mice G1 received no treatment and G2 were treated with Cre. 9th Sacrifice the mice on day one and add Neutrophil 5olation Me. Separate T cells as a mixed lymphocyte band using dia(NIM) and and pooled from 5 mice/group. Results are from 4 replicates of 5 mice/group. Yes, and acts as an average chemotactic coefficient.

In all experiments, non-cyclosporin-treated mice showed a coefficient of 1.76, whereas Mice treated with either 5 or 15 mg/kg cyclosporine-CreL 15% and 33% reduction in chemotactic coefficient, respectively, compared to untreated mice. Indicated. treated with 5 or 15 mg/kg cyclosporine-MLVs The coefficients of mice were 39% and 49% lower than untreated mice, respectively. Repeating this experiment 5 and 15 B/kg tucrosporin-CreL, respectively, 26% and and 37% reduction in chemotactic coefficient, and 5 and 15 mg/kg Suvovorin showed a decrease of 37% and 5196 respectively for MLVs. both fruits In the experiment, 15 mg/kg:/Crossporin-CreL and 5 mg/kg The immunosuppressive activity of cyclosporine-LiLVs was comparable.

CreL alone did not show a significant decrease in PHA-stimulated T cell activity.

Example 10 Tuclosvorin-MLVs and Ciclosporin-CreL or against 5RBC attack to determine whether they have comparable immunosuppressive B cell activity in terms of primary and secondary responses. C57BL/6J mice following either one or two antigen challenges to was treated with either drug form three times.

B cell responses were measured using Jerne plaque analysis and hemagglutination analysis. Response In order to obtain further information about the nature of the antibodies produced in each sample, direct and indirect Both Jerne plaque analyzes were performed to obtain IgM and IgG titers, respectively.

Designed to examine cyclosporine effects on primary responses to 5RBC In the experiment, mice were inoculated with 5RBC on day 0 and The patient was treated with cyclosporine on the same day. In this experiment, Je from 5RBC-challenged control mice rne plaque analysis from direct analysis (1 gM response) to 560 PFU/10' cells. showed 1040 PFU from indirect analysis (combined IgM and IgG responses). Ta. Mice treated with 5 mg/kg cyclosporine-Cre compared to control mice. showed a slightly increased number of plaques in both direct and indirect analysis.

However, mice treated with 5 mg/kg tucrosvorin-MLVs showed a 3.5-fold reduction in plaque numbers in direct analysis when compared to control mice. Ta. This data shows that cyclosporine-MLVs is effective at lower doses of cyclosporine-MLVs. In addition, blood clots from the above-mentioned mice were detected in response to IgM production in the primary response. When looking at concentration titers, the data were similar in some respects to plaque analysis. 5m g/kg of cyclosporine-CreL or cyclosporine-MLVs. compared to 5RBC-challenged control mice, the titer was 1.7-fold and 5-fold, respectively. showed a decrease.

When examining the secondary responses of 5RBC challenge and cyclosporine-treated mice, mice 5RBC1 on the 0th and 4th day and Tsukuro Sports on the 4th, 6th and 8th day. Phosphorus (5 mg/kg) was administered. Cyclosporine-CreL 1gM response were similar to control mice but showed no change in plaque numbers in indirect analysis, and these TgGC against 5RBC in mice? This suggests suppression of answers. However, Mice treated with crosvorin-MLV showed increased 5RBC compared to control mice. showed a decrease in both TgM and IgG. This data also Greater immunosuppression of cyclosporine-MLV compared to crosporin-CreL Shows inhibitory activity.

Example 11 Cyclosporine-NILVs or Tuclos on Mouse B Cell and T Cell Responses To determine the effect of prophylactic treatment of mice with Borin-Cre, mice were given one dose of Pre-prepared with either cyclosporine-MLVs or cyclosporine-CreL. were treated defensively, whereas control mice received no drug treatment prior to 5RBC challenge.

Jerne plaque analysis showed that 5RBC challenged control mice had 560 PFU/10 15 mg/k either 1 or 2 days before antigen challenge. g Cyclosporine-Cre treated mice are similar to those of control mice. It can be seen that the 100% and 500% of the samples show lower (420 and 520, respectively) plaque levels. on the other hand , 15 mg/kg cyclosporine either 1 or 2 days before 5RBC challenge. -Mice treated with MLVs had a lower number of plaques in the analysis compared to controls, each 54% (300) and 50% (280) respectively. 15m 1 or 2 days in advance Mice treated with g/kg cyclosporine-MLVs received an equivalent dose of cyclosporine-MLVs. 28.6 each in plaques compared to mice administered Crosporin-CreL. % and 46% decrease. 3 doses of 5mg/kg cyclosporine - MLVs Mice treated therapeutically (on days 0, 4s and 8) compared to controls. The best plaque reduction was shown with a 3-fold reduction (180) in the number of plaques.

As seen in Table 7, the hemagglutination analysis performed on mouse serum in the aforementioned study We also obtained results.

Antibody titer (sRBC attack) - at dose Chemical treatment (serum collected on day 9) Potency G1: Control (sRBC only) I: l365G 2: 15.0mg/kg Tuclosphorin-CreL (1 day ago ) I:682G 3: 15. Omg/kg Tsucrosphorin-CreL ( 2 days ago) I: 6820 4: 15.0 mg/kg Cyclosporin-AI LVs (1 day ago) I: 256G 5: 15. Omg/kg Tsukuro Hori - MLVs (2 days ago) 1:21G6: 5. Omg/kg cyclosporine -M[, Vs (0th day, 4th day, 8th day) 1:16 In the above experiment, C57BL/6J mice were given 10% 5RB on days 0 and 4. 0.2 ml of CIP was inoculated.

Mice were also treated with 15 mg/kg cyclosporine-CreL or cyclosporine-M. With a single intravenous dose of LVs (either 2 days or 1 day before the sRBC challenge) Processed.

Control mice Gl were inoculated with 5RBC on days 0 and 4, but were treated with cyclosporine. untreated and G6 mice were inoculated with 5RBC and 5 mg/kg cyclo Therapeutically treated with 3 doses (days 0, 4 and 8) of Sporin-MLVs. Ta. All mice were sacrificed on day 9 and blood pooled from 5 mice/group. Hemagglutination analysis was performed on the serum. Results are reported as mean titers of 5 mice/group. Repeat 3 times.

Mice challenged with 5RBC and not given tucrosvorin treatment received I:l365. The titer was shown. 15mg/kg cyclosporine-Cre 1 or 2 days ago Treated mice showed a 2-fold decrease in titer compared to controls. Comparable dosage series Crosporin - 15mg/kg 2 days before compared to Cr eL There was a 32-fold greater decrease in antibody titers in mice treated with MLVs. . Additionally, mice treated with +5 mg/kg cyclosporine-MLVs 2 days prior Mathematically treated with 3 doses of 5 mg/kg cyclosporine-MLVs. had antibody titers comparable to those of mice. This data is for tucrosporin-Cr Unlike comparable doses of eL, a single dose of cyclosporine-MLVs This shows that the immune response against 5RBC can be effectively suppressed by using the 5RBC.

T cells from the above mice were treated with PHA and examined for chemotaxin production. Cyclosporin-CreL and cyclosporin-MLVs treatment when exhausted Chemotactic coefficients in mice were not identical. Non-cyclosporine treated control ma The mice had an average coefficient of 1.46 and received 15 mg/kg 1 day or 2 days ago. Mice treated with cyclosporine-Cre had a coefficient similar to that of control mice. It had However, 15 mg/kg Sisicspoly was administered 1 or 2 days ago. Mice treated with phospho-MLVs were treated with comparable doses of cyclosporine-CreL. showed lower coefficients than treated mice, 23% and 29%, respectively. 15m 2 days ago Mice treated with cyclosporine-MLVs at 5 mg/kg Similar to that of mice treated therapeutically with 3 doses of Rosporin-MLVs showed immunosuppression.

This data shows that both Tm cell responses and B cell responses were positive for cyclosporine-MLV. Although an equivalent dose of cyclosporine-C can be effectively inhibited by prophylactic treatment with s It suggests that remorse cannot be done.

Example ■2 In this experiment, mice were challenged with lipopolysaccharide (LPS), a T-independent antigen, and treated with cyclosporin-MLVs or cyclosporin-Cre. The effect of both agents on murine B cell responses to antigens was determined. Control mice Only LPS was accepted. The results are shown in Table 8.

Antibody titer of mice treated with 3 doses (LPS challenge) G]: Control (Linesome (no cyclosporine) OG2: control (sRBC only) 1:85G 3: CreL 0.15ml of 25% dilution 1:85G5: 5. Omg/kH:/uriUsporin-CreL 1:85G 6:15. Omg/kg Tucrosporin-CreL 1:85G7: 5. Omg/ kg Tsucrosporin-MLVs 1:11G 8: 15. Omg/kg Tuculosporin-MLVs: 13 cases 12 and LPS on days 0 and 40 The B-cell responses of C57BL/6J mice inoculated with 0 mg of T., TVl, and 0 mg were investigated. G 5, G6, C7 and C8 mice were also treated with cyclosporine-CreL or cyclosporin. Porin-MLVs were treated with 3 intravenous doses (days 0, 4 and 8). versus The mice were not treated with either cyclosporine or LPS. LPS to G2 was inoculated but not treated with cyclosporine. G3 was inoculated with LPS and Cr treated with eL and inoculated with 04 LPS and treated with empty liposomes. . All mice were sacrificed on day 9 and serum pooled from 5 mice/group. Hemagglutination analysis was performed. Results are from 3 replicates of 5 mice/group and average Report as average titer.

When mouse serum was analyzed for antibody titers against LPS, non-cyclosporin Treated control mice showed a mean titer of 1:85. Cr eL or Empty - Mice treated with either L also had a titer of I:85 and B cells to LPS. This shows that there is no effect of these substances on the cell response. Similarly, 5 or 15 mg/ Mice treated with kg cyclosporine-Cre also showed increased anti-inflammatory activity compared to control mice. It was shown that there was no decrease in physical strength. However, 5 or 15 mg/kg Mice treated with cyclosporine-MLVs were compared to control mice and cyclosporine-MLVs. each in titer compared to both mice administered comparable doses of CreL. It showed a 7.7-fold and 6.5-fold decrease. This data shows that cyclosporine-MLVs , unlike an equivalent dose of cyclosporine-CreL, B for T-independent antigens. Shows that cell responses can be suppressed. Therefore, this result also indicates that cyclosporine The target of immunosuppression against MLVs is antigen-presenting macrophages; suggests that B cell responses to both T-independent and T-dependent antigens can be influenced by T-independent and T-dependent antigens. Ru.

A one-tailed test was performed to compare cyclosporine-MLVs versus comparable doses of cyclosporine-MLVs. The probability of a difference between the use of CreL was determined. Using each of the three immune assays When cyclosporine ~ MLVs was administered at a comparable dose of cyclosporine -There is a 95% probability that it is significantly more immunosuppressive than Cre L. found.

Although this specification has been disclosed and illustrated in connection with a particular application, the principles involved will be apparent to those skilled in the art. It is obvious that many other applications are possible. The invention therefore resides in the appended claims. The scope is limited only as indicated in the range.

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Claims (1)

[Claims] 1. a (i) neutral phospholipids, (ii) phosphatidylglycerol and dimyly a negatively charged phospholipid selected from stoyl phosphatidic acid, and (i ii) dissolving cyclosporine in an organic solvent to form a solution; b. This melt drying the liquid to form a solid phase; c. Hydrate this solid phase with an aqueous solution to forming a therapeutic preparation for muciclosporin. Preparation of Crosporin therapeutic preparations. 2. (i) neutral phospholipids, (ii) phosphatidylglycerol and dimyris a negatively charged phospholipid selected from toylphosphatidic acid, and (ii i) Ribosomes suspended in an aqueous solution, characterized in that they contain cyclosporin. . 3. (ii) is dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, Sphatidylglycerol, dilaurylphosphatidylglycerol and dimly Claim 1, wherein the compound is selected from the group consisting of stoyl phosphatidic acid. Or method 2. 4. (i) has a lipid moiety composed of 16 or 18 carbon fatty acid chains; Method of request 1 or 2. 5. characterized in that the aqueous solution is a buffer having a pH of 5.5 to 9.5; The method according to claim 1 or 2. 6. Claim in which the ribosome is unilamellar and has a diameter of less than 0.2 μm. Method 1 or 2. 7. (i) has a lipid moiety composed of 16 or 18 carbon fatty acid chains; Method for request 3. 8. characterized in that the aqueous solution is a buffer solution having a pH of 7.0 to 9.5; The method of claim 3. 9. characterized in that the aqueous solution is a buffer having a pH of 5.5 to 9.5; 5. The method of claim 4. 12. Claims that the ribosome is unilamellar and has a diameter of less than 0.2 μm Method of Section 3. 13. Claims that the ribosome is unilamellar and has a diameter of less than 0.2 μm Method of Section 4. 14. Claims that the ribosome is unilamellar and has a diameter of less than 0.2 μm Method of Section 5.